Loss of function BMP4 mutation supports the implication of the BMP/TGF-ß pathway in the etiology of combined pituitary hormone deficiency.

Despite BMP4 signaling being critical to Rathke's pouch induction and maintenance during early stages of pituitary development, its implication in the etiology of combined pituitary hormone deficiency (CPHD) and other clinical presentations of congenital hypopituitarism has not yet been definitely demonstrated. We report here the first CPHD patient with a de novo pathogenic loss-of-function variant in BMP4. A 6-year-old boy, with macrocephaly, myopia/astigmatism, mild psychomotor retardation, anterior pituitary hypoplasia and ectopic posterior pituitary, clinically diagnosed with growth hormone deficiency, and central hypothyroidism, was referred for genetic analysis of CPHD. Targeted NGS analysis with a custom panel (n = 310 genes) identified a novel heterozygous de novo nonsense variant, NM_001202.5:c.794G > A, p.(Trp265*) in BMP4, which introduces a premature stop codon in the BMP4 pro-domain, impairing the transcription of the TGF-ß mature peptide domain. Additional relevant variants in other genes implicated in pituitary development signaling pathways such as SMAD4 and E2F4 (BMP/TGF-pathway), ALMS1 (NOTCH-pathway), and TSHZ1 (Prokineticin-pathway), were also identified. Our results support the implication of the BMP/TGF-ß signaling pathway in the etiology of CPHD and suggest that oligogenic contribution of additional inherited variants may modify the phenotypic expressivity of BMP4 pathogenic variants.

Aldosterone promotes autoimmune damage by enhancing Th17-mediated immunity.

Excessive production of aldosterone leads to the development of hypertension and cardiovascular disease by generating an inflammatory state that can be promoted by T cell immunity. Because nature and intensity of T cell responses is controlled by dendritic cells (DCs), it is important to evaluate whether the function of these cells can be modulated by aldosterone. In this study we show that aldosterone augmented the activation of CD8(+) T cells in a DC-dependent fashion. Consistently, the mineralocorticoid receptor was expressed by DCs, which showed activation of MAPK pathway and secreted IL-6 and TGF-beta in response to aldosterone. In addition, DCs stimulated with aldosterone impose a Th17 phenotype to CD4(+) T cells, which have recently been associated with the promotion of inflammatory and autoimmune diseases. Accordingly, we observed that aldosterone enhances the progression of experimental autoimmune encephalomyelitis, an autoimmune disease promoted by Th17 cells. In addition, blockade of the mineralocorticoid receptor prevented all aldosterone effects on DCs and attenuated experimental autoimmune encephalomyelitis development in aldosterone-treated mice. Our data suggest that modulation of DC function by aldosterone enhances CD8(+) T cell activation and promotes Th17-polarized immune responses, which might contribute to the inflammatory damage leading to hypertension and cardiovascular disease.

Validation of the Clock Drawing Test Scoring Method in older adults with neurocognitive disorder / Validación del método de calificación del Test del Dibujo del Reloj en adultos mayores con trastorno neurocognitivo

Abstract: Introduction: The Clock Drawing Test (CDT) is a widely used instrument for identifying neurocognitive disorders (NCDs) in older adults. However, there is insufficient evidence to determine the best scoring method, since current quantitative methods involve the assignment of numerical values, while qualitative ones do not allow for objectivity in the diagnosis. Parsey & Schmitter-Edgecombe (2011) proposed a scoring scheme which, in addition to providing a score of the patient's performance, permits error analysis, thereby making it possible to identify potential underlying cognitive difficulties. Objective: The purpose of this study was to validate the CDT scoring scheme proposed by Parsey & Schmitter-Edgecombe (2011) for screening for NCDs in Mexican older adults. Method: There were 167 participants: 58 cognitively healthy subjects (CH), 52 with mild neurocognitive disorder (mild-NCD), and 57 with major neurocognitive disorder (major-NCD).The CDT scoring method was compared with the Mini-Mental State Examination (MMSE) and the Montreal Cognitive Assessment in Spanish (MoCA-S). Inter- and intra-observer reliability and construct validity were determined and the sensitivity and specificity of this method were calculated. Results: The X - age was 75 years (SD ± 8 years) and the X - educational attainment was 10.7 years (SD ± 5.2 years). Internal reliability was .750, with an intraclass correlation coefficient of .774. The cut-off point for the CDT in mild-NCD was 14 points (sensitivity: 40%, specificity: 70%) and 12 points for major-NCD (sensitivity: 90%, specificity: 95%).The most frequent errors in the CDT were: graphic, conceptual, spatial, and/or planning difficulties. Discussion and conclusion: This method makes it possibly to quickly and easily explore the cognitive status of the patient. It contains ideal psychometric properties for the detection of patients with major-NCD, in addition to offering the possibility of analyzing performance errors and underlying cognitive difficulties.

Resumen: Introducción: El Test del Dibujo del Reloj (TDR) es un instrumento ampliamente utilizado para identificar trastornos neurocognitivos (TNC) en adultos mayores. Sin embargo, no existe suficiente evidencia para determinar el mejor método para calificarlo, ya que los métodos cuantitativos actuales se abocan a la asignación de valores numéricos, mientras que los cualitativos no permiten objetividad en el diagnóstico. Parsey y Schmitter-Edgecombe (2011) propusieron un método de calificación que, además de proporcionar un puntaje de la ejecución del paciente, permite el análisis de los errores y, con ello, la identificación de las potenciales dificultades cognitivas subyacentes. Objetivo: El objetivo de este estudio fue validar el método de calificación del TDR propuesto por Parsey y Schmitter-Edgecombe (2011) para el tamizaje del TNC en adultos mayores mexicanos. Método: Se contó con 167 participantes: 58 cognitivamente sanos (CS), 52 con trastorno neurocognitivo leve (TNC-leve) y 57 con trastorno neurocognitivo mayor (TNC-mayor). El método de calificación del TDR se comparó con el Examen Mínimo del Estado Mental (MMSE) y la Evaluación Cognitiva de Montreal en español (MoCA-E). Se determinó la confiabilidad inter e intra-observador y la validez de constructo, y se calcularon la sensibilidad y la especificidad de este método. Resultados: La X - de edad fue de 75 años (DE ± 8 años) y la X - de escolaridad fue de 10.7 años (DE ± 5.2 años). La confiabilidad interna fue de .750, con un coeficiente de correlación intraclase de .774. El punto de corte para el TDR en TNC-leve fue de 14 puntos (sensibilidad: 40%, especificidad: 70%) y 12 puntos para TNC-mayor (sensibilidad: 90%, especificidad: 95%). Los errores más frecuentes en el TDR fueron: dificultades gráficas, conceptuales y espaciales, y/o de planeación. Discusión y conclusión: Este método permite explorar breve y ágilmente el estado cognitivo del paciente y posee propiedades psicométricas ideales para la detección de pacientes con TNC-mayor, además de ofrecer la posibilidad de analizar los errores que presentan en el desempeño y las dificultades cognitivas subyacentes.

Immune complex-induced enhancement of bacterial antigen presentation requires Fcgamma receptor III expression on dendritic cells.

Dendritic cells (DCs) are capable of initiating adaptive immune responses against infectious agents by presenting pathogen-derived antigens on MHC molecules to naïve T cells. Because of their key role in priming adaptive immunity, it is expected that interfering with DC function would be advantageous to the pathogen. We have previously shown that Salmonella enterica serovar Typhimurium (ST), is able to survive inside DCs and interfere with their function by avoiding activation of bacteria-specific T cells. In contrast, when ST is targeted to Fcgamma receptors on the DC surface, bacteria are degraded and their antigens presented to T cells. However, the specific Fcgamma receptor responsible of restoring presentation of antigens remains unknown. Here, we show that IgG-coated ST was targeted to lysosomes and degraded and its antigens presented on MHC molecules only when the low-affinity activating FcgammaRIII was expressed on DCs. FcgammaRIII-mediated enhancement of Ag presentation led to a robust activation of T cells specific for bacteria-expressed antigens. Laser confocal and electron microscopy analyses revealed that IgG-coated ST was rerouted to the lysosomal pathway through an FcgammaRIII-dependent mechanism. PI-3K activity was required for this process, because specific inhibitors promoted the survival of IgG-coated ST inside DCs and prevented DCs from activating bacteria-specific T cells. Our data suggest that the DC capacity to efficiently activate T cells upon capturing IgG-coated virulent bacteria is mediated by FcgammaRIII and requires PI-3K activity.

Evaluación de la capacidad de desarrollo in vitro de ovocitos bovinos provenientes de vacas con predominancia fenotípica Bos taurus y Bos indicus / Evaluation of in vitro development capacity of bovine oocytes obtained from predominantly Bos taurus and Bos indicus cows

El objetivo de este estudio fue evaluar la capacidad de desarrollo in vitro de ovocitos bovinos de vacas mestizas B. taurus y B. indicus. Los ovocitos fueron recuperados de ovarios de hembras bovinas provenientes de un matadero comercial. Para la obtención de los complejos cumulus-ovocitos (CCO) se realizó la técnica de slicing, seleccionando los ovocitos que tenían al menos una capa de células del cumulus y un citoplasma homogéneo. Los ovocitos seleccionados fueron madurados y fecundados in vitro (MIV-FIV). Se utilizó semen de un toro Brahman puro (B. indicus). Para la evaluación de la MIV y FIV todos los ovocitos se fijaron por al menos 24 h a 4°C en solución metanol-ácido acético (3:1) y teñidos con aceto-orceína al 1,1%. La tasa de maduración de ovocitos de vacas con predominancia fenotípica B. indicus fue del 66,17% mientras que las vacas con predominancia fenotípica B. taurus alcanzaron un 50,94% (P>0,05). En cuanto a la tasa de fecundación se obtuvo un 14,28 y 35,72% de ovocitos penetrados normalmente y anormales, respectivamente, para el grupo de ovocitos con predominancia fenotípica B. indicus. Mientras que para vacas con predominancia fenotípica B. taurus, un 10,22% correspondió a ovocitos penetrados normales y 19,31% de ovocitos penetrados anormales, sin encontrar diferencias estadísticamente significativas en ambos grupos. Los presentes resultados, tanto para la progresión meiótica como para las tasas de fecundación, indican que los ovocitos de vacas mestizas con predominancia fenotípica B. indicus son más competentes en las primeras etapas de desarrollo in vitro que los ovocitos de vacas mestizas con predominancia fenotípica B. taurus.

The aim of this study was to evaluate the in vitro development capacity of bovine oocytes from crossbred B. taurus and B. indicus cows. Oocytes from bovine cows were collected from commercial slaughterhouse. The cumulus-oocyte complex (COC) ovaries were obtained by Slicing technique, selecting those oocytes that had 2 to 3 layers of cumulus cells and homogeneous cytoplasm. After selection oocytes proceed with maturation (IVM) and fertilization in vitro (IVF). It used semen from a pure Brahman bull (B. indicus). For the assessment of IVM as for IVF oocytes were fixed for about 24 hours at 4°C in methanol-acetic acid (3:1) solution and stained with 1.1% aceto-orcein. The maturation rate of oocytes from cows with B. indicus phenotypic predominance was 66.17%, whereas cows with B. taurus phenotypic predominance 50.94% (P>0.05). Fertilization rate obtained in B. indicus phenotypic predominance group was 14.28% of oocytes normal penetrated and abnormal penetrated 35.72%, for cows with a phenotypic predominance B. taurus oocytes normal penetrated were 10.22% and 19.31% of abnormal oocytes penetrated. In conclusion, the present results indicate that oocytes from cows with phenotypic predominance B. indicus are more competent in the early stages of development in vitro than oocytes from cows with phenotypic predominance B. taurus.

Roles of oxidative stress and glutathione depletion in JP-8 jet fuel-induced apoptosis in rat lung epithelial cells.

The toxic jet fuel JP-8 induces morphological and biochemical changes characteristic of apoptosis in rat lung epithelial (RLE-6TN) cells. The mechanism of JP-8 toxicity in these cells was further investigated in an attempt to identify potential therapeutic interventions. Given that oxidative stress and changes in the concentrations of endogenous antioxidants, such as glutathione (GSH), have been associated with the cellular damage elicited by numerous toxicants, the possibility that JP-8 induces cellular oxidative stress was investigated. Experimentally induced depletion of intracellular GSH or exposure of cells to a low concentration of H(2)O(2) markedly enhanced JP-8-induced cell death. A significant reduction in intracellular concentrations of GSH was noted in RLE-6TN cells shortly after exposure to JP-8. Furthermore, JP-8 induced the generation of reactive oxygen species (ROS) in RLE-6TN cells. Consistent with the notion that JP-8 toxicity is mediated by generation of ROS and depletion of intracellular GSH, JP-8-induced cell death was inhibited by exogenous GSH or the thiol-containing antioxidant N-acetyl-cysteine. This protective effect was associated with marked inhibition of both the activation of caspase-3 and the loss of the mitochondrial membrane potential induced by JP-8. Inhibition of the JP-8-induced activation of poly(ADP-ribose) polymerase by 3-aminobenzamide did not protect cells against JP-8 toxicity. Together, these results indicate that thiol antioxidants are highly effective in rescuing cells from JP-8-induced cell death and that they may provide a basis for new therapeutic approaches to counteract JP-8 toxicity.

Regulation of DNAS1L3 endonuclease activity by poly(ADP-ribosyl)ation during etoposide-induced apoptosis. Role of poly(ADP-ribose) polymerase-1 cleavage in endonuclease activation.

Several endonucleases are implicated in the internucleosomal DNA fragmentation associated with apoptosis. The human Ca2+- and Mg2+-dependent endonuclease DNAS1L3 is inhibited by poly(ADP-ribosyl)ation in vitro, and its activation during apoptosis shows a time course similar to that of the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). The role of the cleavage and consequent inactivation of PARP-1 by caspase-3 in the activation of DNAS1L3 has now been investigated further both in vitro and in vivo. In an in vitro system based on purified recombinant proteins and NAD, caspase-3 prevented the inhibition of DNAS1L3 endonuclease activity by wild-type PARP-1 but not that induced by a caspase-3-resistant PARP-1 mutant. The induction by etoposide of apoptosis in human osteosarcoma cells (which were shown not to express endogenous DNAS1L3) was accompanied by internucleosomal DNA fragmentation only after transfection of the cells with a plasmid encoding DNAS1L3. This DNA fragmentation in etoposide-treated cells was blocked by 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, an inhibitor of intracellular Ca2+ release. Expression of the endonuclease subunit of DNA fragmentation factor (DFF40) and cleavage of its inhibitor, DFF45, were not sufficient to cause internucleosomal DNA fragmentation in osteosarcoma cells during etoposide-induced apoptosis. Coexpression of caspase-3-resistant PARP-1 mutant with DNAS1L3 in osteosarcoma cells blocked etoposide-induced internucleosomal DNA fragmentation and resulted in persistent poly(ADP-ribosyl)ation of DNAS1L3; it did not, however, prevent the activation of caspase-3 and the consequent cleavage of endogenous PARP-1. These results indicate that PARP-1 cleavage during apoptosis is not simply required to prevent excessive depletion of NAD and ATP but is also necessary to release DNAS1L3 from poly(ADP-ribosyl)ation-mediated inhibition.

Estudio estructural del huso meiótico de ovocitos bovinos vitrificados / Structural study of meiotic spindle of vitrified bovine oocytes

La verificación de ovocitos bovinos se ha venido utilizando como uno de los protocolos de criopreservación más prometedores. Se ha expresado el temor por los efectos que puede causar la utilización de aditivos crioprotectores sobre la integridad estructural de los ovocitos. En este trabajo, los experimentos fueron conducidos para evaluar e efecto de vitrificación sobre la estructura del huso meiótio y la capacidad de desarrollo de ovocitos bovinos. Para ello, ovocitos acabados de loberar de sus folículos y madurados in vitro fueron expuestos a temperatura ambiente a las soluciones vitrificadoras (etilendlicol y sacarosa), también fueron vitrificados ovocitos inmaduros y madurados in vitro. Los ovocitos fueron teñidos in toto, para evaluar la maduración nuclear y a través de la tinción inmunocitoquímica fueron estudiadas las alteraciones estructurales del huso mieótico. Los ovocitos en estadio de VG, son más sensibles al efecto tóxico causado por los agentes crioprotectores a temperatura ambiente y al proceso de criopreservación, valorados mediante la progresión meiótica y el análisis inmunocitoquímico del huso meiótico. La anomalía más frecuentemente observada fue la ausencia del huso meiótico. La estructura del huso meiótico de ovocitos vitrificados MIV, es más resistentes al daño crioinducido, que los ovocitos vitrificados en estadio de VG. Se recomienda la vitrificación de ovocitos bovinos madurados in vitro.

Bovine oocytes vitrification has been used as one of the most promising cryopreservation protocols. Fear of the effects of cryoprotectant additives on the oocytes structural integrity have been posed. In this study, trials were conducted to evaluate the effects of vitrification over meiotic spindle structure and development capability in bovine oocytes. Based in this evidence, oocytes recently released from their follicles and in vitro matured were exposed at room temperature to vitrifying solutions (ethylenglicol and sucrose), also immature and in vitro matured oocytes were vitrified. Oocytes were stained in toto to assess nuclear maturation stage and meiotic spindle structural alterations were studied by immunocytochemistry. Oocytes at GV stage are more sensitive to the toxic effects caused by cryoprotectant agents at room temperature and to the cryopreservation procedure, assessed through meiotic progression and immunocytochemical analysis of meiotic spindle. The most frequently encountered anomaly was absence of meiotic spindle. Meiotic spindle structure in vitrified and IVM oocytes endure better the cryoinduced injuries compared with oocytes vitrified the GV stage. It is recommend the vitrification of in vitro matured bovine oocytes.